Presentation title Micro LC/MS: Miniaturizing LC for LC/MS ...

1 © 2011 AB SCIEX

Presentation title Name of Speaker and Identification, Location, Date

Micro LC/MS: Miniaturizing LC for LC/MS applications in bioanalysis Tina Settineri, Ph.D. Director, HPLC Products

2 © 2011 AB SCIEX

What is Microflow LC?

0.5 mm

area = 18 area = 1area = 85

0.5 mm

area = 18 area = 1area = 85

• MicroLC: HPLC using columns with inner diameter (ID) ≤ 1.0 mm. •  Typical Flow Rates: 5-200µL/min

3 © 2011 AB SCIEX

�  Improve throughput

� Use less sample

�  Improve sensitivity (LOQ) of LC/MS

�  Improve LC and MS uptime

�  Save money spent on mobile phases

Why Miniaturize to Micro flow UHPLC for LC/MS Bioanalysis?

4 © 2011 AB SCIEX

ekspertTM microLC 200 micro flow UHPLC

•  Microflow UHPLC (10,000 psi) •  System delay (void) volume: 1 – 3 µL •  Total system volume: ~ 4.5 µL

•  Supports 1.0, 0.5, and 0.3 mm ID columns •  Flow rates 5 – 200 µL/min •  Programmable injection - from 15 nL (metered)

to 10 µL (without changing loop) •  Close coupling with MS source

-  Small volume hybrid electrodes for Turbo VTM source

Compact, high-throughput UHPLC system for LC/MS

5 © 2011 AB SCIEX

Microfluidic Flow Control™ (MFC) Unique pumping system - flow control at nano/micro low flow rates •  Direct flow rate feedback to a rapidly adjustable

pressure source

•  Maintains flow rate independent of backpressure

•  Enables highly reproducible and accurate analytical separations

•  Single-stroke-per-run design for reliability and performance

0 60 120 180 240 300395

396

397

398

399

400

401

402

Flow

Rat

e (n

L/m

in)

Time (sec)

1 nL/min step

2 nL/min step

Electronically Controlled Pressure Source

Flow Meter

Control Processor

6 © 2011 AB SCIEX

Flow lamination to decrease diffusion length by 2x and hence improve mixing by 4x

A

B B A

Pumps

Purge Valves

Filters

Pressure Transducers

Pb

A

B Pc

Pa

Flow Module (restrictors)

Pumping system - Unique 3 Layer Mixing Design Significantly Decreases mixing & delay time

7 © 2011 AB SCIEX

Decreasing diffusion length, x, decreases time required for mixing by x2

Required mixing time t (or mixer length)

Two thick streams

Three thinner streams

t = x2

x

x’

1 2 3 4

•  In a two layer mixer, the longest diffusion distance needed for full mixing is the tube diameter

•  In a three layer the max diffusion distance needed to travel is only the tube radius

Pumping system - Unique 3 Layer Mixing Design Significantly Decreases mixing & delay time

8 © 2011 AB SCIEX

Improved Throughput with Low Delay Times: Micro Flow UHPLC – actual gradient = programmed gradient High Flow UHPLC – actual gradient ≠ programmed gradient - especially with fast separations

- reduces inject-to-inject times - allows a wider range of compounds eluted during gradient - improves accuracy with fast gradients

Time

Programmed gradient

Actual microLC gradient

Actual analytical UHPLC gradient

Column Diameter (mm)

Delay Volume/ Void Volume

UHPLC 2.1 1.0

MicroLC 0.5 0.13

Improving Throughput with Micro Flow UHPLC:

9 © 2011 AB SCIEX

Improved Throughput with Low Delay Times:

Traditional UHPLC Microflow UHPLC

Flow Rate 1.5 mL/min 0.060 mL/min

Column Diameter 2.1 mm 0.50 mm

Mobile Phase / Solvent Waste Volume / run

1925 µL 66 µL

Solvent Used / 1200 injections 2.31 L 2 mL

10 20 30 40 50 60 70

-500

0

500

1000

1500

2000

14

13

12

11

109

8

7

6

5

4

2

mAU

Seconds

1

3

10 20 30 40 50 60 70

-200

0

200

400

600

800

14

13

1211

10

9

8

7

65

4

3

2

mAU

Seconds

1

Analytical UHPLC, 77 sec Microflow UHPLC, 48 sec

10 © 2011 AB SCIEX

RT: 0.00 - 2.50

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative

Abunda

nce

1.16

1.98

1.44

1.44

1.45

1.43

1.671.66

1.460.89 0.89 1.65

1.40 1.47

1.481.21 1.70 2.05 2.452.310.84 1.010.710.58 1.950.03 0.14 0.39

NL:2.76E6TIC MS RT9019-24

RT: 0.00 - 1.00

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

0.82

0.820.48

0.48

0.82

0.530.83

0.56

0.610.47 0.830.81

0.61

0.83

0.62 0.640.47 0.81 0.840.840.65 0.810.75 0.86 0.920.470.350.120.07 0.19 0.310.25 0.37

NL:2.40E7TIC MS Dev-10

10

Traditional LC: Multiple Analytes

Low Flow: Multiple Analytes

Exceptional Separation Equivalent Sensitivity using 10-20x less sample 1/3 the overall run time

Improved Throughput with Low Delay Times:

Data courtesy of Rob Turner, CoMentis

11 © 2011 AB SCIEX 11

�  Excellent separation with 0.5 to 1 minute chromatography

�  Solvent usage decreased from ~ 2-3 Liters/week to ~ 20 mls/week

�  Reduced sample injection size from 10-20 µLs to 1-2 µLs without a loss in sensitivity, reduces sample required, enabling multiple sampling and Single animal PK/PD even with rodent species

�  Extended linear range observed

�  Decrease in matrix effect observed

Summary - MicroLC System Incorporation at CoMentis

Slide courtesy of Rob Turner, CoMentis

12 © 2011 AB SCIEX

Electrode Dispersion Comparison (10 µL/min)

• Peak dispersion as observed in a flow injection experiment at 10 µL/min for four ESI electrodes with different internal diameter sizes.

• The orange and blue lines illustrate the peak dispersion improvement utilizing the new smaller ID hybrid electrodes

13 © 2011 AB SCIEX

600µL/min Standard electrode

800µL/min Standard electrode

38µL/min 65µm ID

80µL/min 65µm ID

38µL/min 50µm ID

80µL/min 50µm ID

Example Data: 1 ng/mL, 1 µL injection, same gradient Improvement in peak width at base with smaller ID electrodes

Shimadzu system - high flow ekspert™ microLC 200 system - micro flow

1.8 sec 1.8 sec 1.5 sec

2.1 sec 2.4 sec 1.5 sec

14 © 2011 AB SCIEX

�  Two new smaller ID electrodes for use in the TurboV™ source –  25 µm ID - ideal for 0.3 mm columns (5-20 µL/min) –  50 µm ID - ideal for 0.5 mm columns (20-100 µL/min)

�  Hybrid PEEKSIL/stainless steel tip electrodes

�  Easy to install by replacing standard electrode

25 µm ID 50 µm ID

1 cm

Column very close to source

MicroLC system uses new hybrid electrodes

15 © 2011 AB SCIEX

Improved Chromatography with hybrid electrodes & ekspert™ microLC 200 System

1A9 Peptide 1

2B7 Peptide 2

2B7 Peptide 1

1A6 Peptide 2

Endogenous

1A6 Peptide 1 1A1 Peptide 1

Endogenous

1A9 Peptide 1

2B7 Peptide 2

2B7 Peptide 1

1A6 Peptide 2

1A6 Peptide 1

1A1 Peptide 1

Endogenous

Endogenous

1A1 peptide 2

Data courtesy of Pharmacadence (in collaboration with Merck)

With hybrid electrode

With hybrid electrode

Old style larger ID electrode

Old style larger ID electrode

16 © 2011 AB SCIEX

0.25 µL sample pickup and injection with 0.6% RSD (n=3)

y = 3E+06x - 346238R2 = 0.9973

0

1

2

3

4

5

6

0 0.5 1 1.5 2

Millions

Sample Volume (µL)

Peak

are

a (c

ount

s)

Verapamil Peak Area (counts)

Linear fit (blank injection excluded)

Use less sample: AS/valve design enables very small volume injections with low RSDs

10 ng/mL Verapamil, 53 sec cycle time 5-90% B gradient in 30 seconds, 0.5 x 50 mm Halo C18 column.

Sample Vol (µL) %RSD, n=3 0.25 0.643407

0.50 1.109915

0.75 0.891552

1.0 0.630341

1.25 1.750287

1.50 1.474698

1.75 1.229087

2.0 1.691631

17 © 2011 AB SCIEX

% Sampling Efficiency, entire flow range, 0.05-500 µL/min

Flow Rate (µL/min)

0

5

10

15

20

25

0.5 100 200 300 400 500 5

20 60

Sampling Efficiency %

0.05

120 250

22.0%

% Sampling Efficiency, high flow range only, 5-500 µL/min 2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8 Sampling Efficiency %

0.20% 0.27%

0.40% 0.50%

0.80%

1.50%

7.5x

110x

Covey,T.R.; Thomson B.A.; , Schneider, B.S. “Atmospheric Pressure Ion Sources,” Mass Spec. Reviews, 2009, 00, 1-29. Covey TR, Schneider BB, Kovarik, Corr, Javahari et al. “”The Central Analytical Figures of Merit of ESI, MALDI, and APCI.” In:Cole RB, ed. Electrospray and MALDI mass spectrometry. 2010, Chapter 13. Hoboken: John Wiley & Sons, Inc.

Sampling Efficiency vs. Flow Rate in ESI - Turbo V™ Source API 5000 System

18 © 2011 AB SCIEX

High Flow Steroid Method vs. Micro Flow Method �  Equal sample volume & concentration was injected on column for both methods

�  Peak areas increased by about 10X on average and S/N improved by 4X

XIC of +MRM (8 pairs): 347.200/293.200 Da ID: Corticosterone_57 from Sample 40 (std 3, 11,111.11 pg_mL) of InertSustain 2.1x... Max. 1.3e4 cps.

1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

1.0e4

1.1e4

1.2e4

1.3e4

1.4e4

1.5e4

1.6e4

1.7e4

1.8e41.8e4

Inten

sity, c

ps

XIC of +MRM (8 pairs): 347.200/293.200 Da from Sample 43 (std 3, 11,111.11 pg_mL) of 0.3x100mm Sustain calcurve.wiff (Tur... Max. 8.2e4 cps.

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

Inten

sity, c

ps

1.4e5

1.8e4

600 µL/min 2 µL inj.

10 µL/min 2 µL inj.

Signal to Noise Comparison of MicroLC vs. High Flow Steroid Method. Data points and replicates which produced accuracies of 100% ± 10% were used to calculate average values in the table. Ratios are expressed as low flow/high flow.

Compound Conc. (pg) Area Ratio

Peak Height Ratio S/N Ratio

Corticosterone 2.47 10.5 6.0 6.0

7.41 11.0 5.6 5.6

22.22 11.6 5.3 5.2

66.67 11.5 5.8 5.6

Average 11.2 5.7 5.6

%CV 4.4 5.6 5.9

Aldosterone 7.41 9.2 7.6 3.9

22.22 8.8 6.7 3.2

66.67 8.3 6.5 2.9

Average 8.8 6.9 3.3

%CV 5.2 8.3 16.2

19 © 2011 AB SCIEX

LC-MS/MS Overlayed Chromatograms (HPLC vs. MFLC/QTRAP® 5500 System Bioanalysis of Methotrexate (400 ng/mL) from Human Plasma

Similar linear velocities Same stationary phase Prontosil, C18, 3 µm

0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 Time, min

5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 4.0e5 4.5e5 5.0e5 5.5e5 6.0e5 6.5e5 7.0e5 7.5e5

Intensity, cps

HPLC – 700 µL/min, 2.0 mm ID x 50 mm Injection volume = 2 µL

MFLC – 35 µL/min, 0.5 mm ID x 50 mm, Injection volume = 2 µL

9.6x Signal Increase!

Data courtesy of Alturas Analytics, Inc.

20 © 2011 AB SCIEX

Overall LLOQ Sensitivity Compared to Traditional Analytical LC Method

Column ID (mm)

Flow rate (µl/min)

Max Injection

Volume (µl) Avg Area % Diff.

2.0 400 40 1.27E06 N/A

0.3 10 10 9.37E05 -26

Data produced from injection of propranolol on QTRAP® 5500

Data courtesy of Alturas Analytics, Inc.

21 © 2011 AB SCIEX

�  Improve throughput Very low delay volume of system enable ultrafast gradients for LC/MS withough compromising chromatography

� Use less sample – inject smaller volumes, minimize waste Injection system enables sub 1 µL injections and minimal sample wasted

�  Improve sensitivity (LOQ) for LC/MS Improved ionization efficiency translates & improved chromatographic peak capacity translates into improved sensitivity & S/N

�  Improve LC and MS Uptime 1-stroke per run minimizes LC maintanence, lower flow rates & injection volumes provide reduced solvent load on MS

�  Save Money spent on mobile phases Up to 95% lower mobile phase costs, including storage and disposal

Miniaturization to Micro flow UHPLC

22 © 2011 AB SCIEX

Trademarks/Licensing

For Research Use Only. Not for use in diagnostic procedures.

The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

© 2012 AB SCIEX.

23 © 2011 AB SCIEX

Robustness for DMPK DMPK - Robustness Test – 10 µL/min, 5+ days, 750+ injections

Peak Area Response Ratio of Imipramine vs. Clomipramine spiked into rat plasma

0.00.20.40.60.81.01.21.41.6

10/31 11/1 11/2 11/3 11/4 11/5 11/6 11/7

Are

a R

espo

nse

Rat

io

Date of acquisitionXIC of +... Max. 2.1e5 cps.

5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

Intensity, cp

s

1.06

XIC of +... Max. 2.1e5 cps.

5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

Intensity, cp

s

1.18


5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

Intensity, cp

s

1.27


5Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

Intensity, cp

s

1.31

Day 1 Day 3 Day 4 Day 5

2.1e5 2.1e5 2.0e5 2.2e5

No loss in signal intensity or chromatographic performance

observed across the 750+ replicate injections.

0.3 mm ID × 50 mm ChromXP™ HALO C18 column

Avg. response ratio = 1.3, Std Dev. = 0.05 3.8% SD over the entire analysis

24 © 2011 AB SCIEX

Inject time = Required volume

Measured flow rate

•  Technique for delivering nanovolume injections

•  Programmable / variable injection volumes

•  Excellent precision / linearity

•  Multiple injections from single A/S sample load allows method development with a small volume of sample

•  Faster method development by more efficient system utilization

y = 1428.7 x - 238256R2 = 0.9979

0.E+00

2.E+06

4.E+06

6.E+06

8.E+06

1.E+07

1.E+07

1.E+07

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000

Injected Volume (uL)

Pea

k A

rea

Metered, ‘Time-slice’ Injections for nanovolume injections to save sample for microLC

0 2 4 6 8 10

R2=0.9998

Injected Volume (nL) 0 500 1000

Pea

k A

rea

25 © 2011 AB SCIEX

Eksigent UHPLC Product Portfolio

�  From Analytical Flow to Micro Flow to Nano Flow LC

ekspert™ ultraLC 100 & 100-XL systems The only analytical flow UHPLC systems designed for use with AB SCIEX mass spectrometers

ekspert™ microLC 200 system The world’s only dedicated micro-UHPLC designed to deliver faster separations and improved sensitivity for LC/MS

NanoLC-Ultra® & cHiPLC system High Performance splitless flow nano-UHPLC coupled with the plug and play, high reproducibility cHiPLC system

ExpressLC® – UV System High Performance microflow UHPLC system

26 © 2011 AB SCIEX

Delay volume effect – high flow vs. microflow 25 µm electrode, 2 µL inj. (Triazine Pesticide Mixture – 10 µL/min flowrate) Chromatogram for 0.5 X 50 mm Halo™ C18 column run with the conventional LC system optimized for low flow Chromatogram for 0.5 X 50 mm Halo™ C18 column run with the ekspert™ microLC 200 MS: AB SCIEX 3200 QTRAP®

27 © 2011 AB SCIEX

Tubing, flow rates & electrodes for various column diameters

Column ID (mm)

Flow rate range (µL/min)

Connection tubing ID (µm)

Recommended ESI electrode

2.1 200-1000 ~125 (0.005”) Standard TurboV electrode

1.0 50-200 75 pre-column 50 post-column

65µm ID stainless steel or 50µm ID PEEKsil/stainless steel

0.5 10-50 50 pre-column 50 post-column

50µm ID PEEKsil/stainless steel

0.3 4-20 50 pre- column ≤50 post-column

25µm or 50µm ID hybrid PEEKsil/stainless steel

Presentation title Micro LC/MS: Miniaturizing LC for LC/MS ...

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